Method of fixing biomolecule on metal support

ABSTRACT

A solution of a nucleic acid is spotted on a metal carrier consisting of a metal selected from Groups I, II, III, IV, V, VI, and VII of second to seventh periods and transition elements in a periodic table, or of an alloy containing the metal, and the solution is dried. Then, the nucleic acid is immobilized on the carrier spotted with the solution by irradiating the carrier with an ultraviolet ray containing a component having a wavelength of 280 nm, of which irradiation dose is 100 mJ/cm 2  or more.

TECHNICAL FIELD

The present invention relates to a method of immobilizing a biomoleculesuch as a nucleic acid on a carrier. The method of the present inventionis useful for operations of analysis of nucleic acids based onhybridization and so forth.

BACKGROUND ART

In analyses of nucleic acids based on hybridization, immunoassays and soforth, techniques of immobilizing nucleic acids or proteins on carrierssuch as membranes and plates have conventionally been utilized. As suchmethods of immobilizing biomolecules, the following methods are knownfor nucleic acids:

(1) A method of chemically binding a nucleic acid introduced with amodification group, such as immobilization by a disulfide bond between anucleic acid having a thiol group at the 5′ end and a bead-like basematerial having thiol groups (P. J. R. Day, P. S. Flora, J. E. Fox, M.R. Walker, Biochem. J., 278, 735-740 (1991));

(2) A method of immobilizing a nucleic acid by adsorption on a carriersuch as nitrocellulose, nylon membrane, or glass coated with a cationpolymer such as poly-L-Lysine through ultraviolet (UV) irradiation orheat treatment (J. Sambrook, E. F. Fritsch and T. Maniatis, MolecularCloning, Cold Spring Harbor Laboratory Press, Second Edition, pages2.109-2.113 and pages 9.34-9.46, JP 10-503841 A);

(3) A method of immobilizing nucleic acid on the basis of physicaladsorption obtained by injecting the nucleic acid into wells of amicroplate treated with a polylysine solution and heating the plate at37° C. (G. C. N. Parry and A. D. B. Malcolm, Biochem. Soc. Trans., 17,230-231 (1989));

(4) A method comprising synthesizing DNA on a base material by usingnucleotides which bonded to the base material (International PublicationPamphlet No. 97/10365 (WO97/10365)); and

(5) A method of immobilizing a nucleic acid on a base material such asglass carrying a polymer compound having carbodiimide groups (JP 8-23975A).

However, the method of (1) requires an extremely special apparatus andregents. Furthermore, in the methods of (2) and (3), nucleic acids aredropped off from the carriers during the hybridization, in particular,in operation processes, and thus detection sensitivity may be reduced,or reproducibility cannot be obtained. Furthermore, those methods sufferfrom further drawback, that is, although a long nucleic acid can beimmobilized, a short nucleic acid of about 50-mer or shorter such asoligomers cannot be efficiently immobilized. In those methods, the UVdose is about several tens mJ/cm². Furthermore, the method of (4) alsorequires an extremely special apparatus and regents for synthesizing DNAon a base material, and the nucleic acid that can be synthesized islimited to about 25-mer or shorter. Moreover, in the method of (5), thematerial of the base material is limited, and a surface coating step isrequired.

DISCLOSURE OF THE INVENTION

In view of the aforementioned technical situations of the conventionaltechniques, an object of the present invention is to provide a method ofconveniently and efficiently immobilizing a biomolecule such as anucleic acid, in particular, a nucleic acid of a short chain length, ona carrier.

The inventors of the present invention have conducted various researchesin order to achieve the aforementioned object. As a result, they havefound that a nucleic acid can be efficiently immobilized on a carrier byspotting a solution of the nucleic acid on a metallic carrier and thenirradiating the carrier with an ultraviolet ray, and have accomplishedthe present invention.

Thus, the present invention provides the following.

(1) A method of immobilizing a biomolecule on a carrier, including thesteps of: spotting a solution of the biomolecule on the carrier; andirradiating the carrier spotted with the solution of the biomoleculewith an ultraviolet ray containing a component having a wavelength of280 nm, in which the carrier is made of a metal.

(2) The method according to (1), in which the ultraviolet ray contains acomponent having a wavelength of 220 to 300 nm.

(3) The method according to (1) or (2), in which the metal is a metalselected from Groups I, II, III, IV, V, VI, and VII of second to seventhperiods and transition elements in a periodic table, or an alloycontaining any of these metal.

(4) The method according to any one of (1) to (3), in which irradiationdose of the ultraviolet ray is 100 mJ/cm² or more.

(5) The method according to any one of (1) to (4), in which thebiomolecule is selected from a nucleic acid, protein, saccharide,antigen, antibody, peptide, and enzyme.

(6) A method of producing a biomolecule-immobilized carrier in which abiomolecule is immobilized on a carrier, including the steps of:spotting a solution of the biomolecule on the carrier; and irradiatingthe carrier spotted with the solution of the biomolecule with anultraviolet ray containing a component having a wavelength of 280 nm toimmobilize the biomolecule on the carrier.

(7) The method according to (6), in which the ultraviolet ray contains acomponent having a wavelength of 220 to 300 nm.

(8) The method according to (6) or (7), in which the biomolecule is anucleic acid, and the nucleic acid-immobilized carrier is used foranalysis of the nucleic acid by hybridization.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 (photograph) shows the result of hybridization using theoligonucleotide-immobilized plate produced in the example.

The dotted line represents the regions on which the oligonucleotideswere immobilized, and the regions on which 1×TE buffer solution wasspotted as a control in Example 1 and Comparative Example 1.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained in detail.

A carrier used in the present invention is intended to immobilize abiomolecule, and is made of a metal. The metal is not particularlylimited as long as it can immobilize a biomolecule by ultraviolet rayirradiation. Preferable examples thereof include a metal selected fromGroups I, II, III, IV, V, VI, and VII of second to seventh periods andtransition elements in a periodic table, and an alloy containing each ofthese metal.

Particularly preferable examples of the metal selected from Groups I,II, III, IV, V, VI, and VII of second to seventh periods and transitionelements in the periodic table include aluminum, titanium, platinum,tungsten, molybdenum, gold, copper, and nickel.

Specific examples of the alloy include nickel silver (component: Cu, Ni,Zn), brass (component: Cu, Zn), bronze (component: Cu, Be), Monel(component: Cu, Ni, Fe, Mn), a nickel-cobalt alloy (component: Ni, Co),a nickel-chromium alloy (component: Ni, Cr), a cobalt alloy (component:Co, Ni, Cr), stainless steel (component: Ni, Cr, Fe), silver tungsten(component: Ag, W), P titanium (component: Ti, V, Al), αβ titanium(component: Ti, V, Al), an NT alloy (component: Ti, Ni), an aluminumalloy (component: Al, Cu, Mg, Si, Mn, Zn), duralumin (component: Al, Cu,Si, Fe, Mn, Mg, Zn), a magnesium alloy (component: Mg, Al, Zn), K24(component: Au), K18 (component: Au, Ag, Cu), beryllium copper(component: Cu, Be), cast iron (component: Fe, Mn, S, C), carbon steel(component: Fe, C, Si, Mn, P, S), bronze casting (component: Cu, Sn, Zn,Pb), phosphor bronze casting (component: Cu, Zn, P), brass casting(component: Cu, Zn, Pb), manganese brass (component: Cu, Zn, Mn, Fe,Al), silzin bronze casting (component: Cu, Si, Zn), aluminum bronzecasting (component: Cu, Al, Fe, Ni, Mn), elinvar (component: Ni, Cr,Mn), elinvar extra (component: Ni, Cr, Co, Mn), invar (component: Ni,Fe), super invar (component: Fe, Ni, Co), stainless invar (component:Fe, Co, Cr), Malottes (component: Sn, Bi, Pb), Lipowitz (component: Sn,Bi, Pb, Cd), Wood's (component: Sn, Bi, Pb, Cd), manganin (component:Cu, Mn, Ni, Fe), izabellin (component: Cu, Mn, Al), constantan(component: Cu, Ni), arcless (component: Fe, Cr, Al), kanthal(component: Cr, Fe, Al, Co), alumel (component: Ni, Al), a magneticmaterial (a material containing a ferromagnetic transition element suchas Fe, Ni, or Co), permalloy (component: Fe, Ni), alpalm (component: Fe,Al), ferrite (complex oxide having Fe₂O₃ as a main component), sendust(component: Fe, Si, Al), super sendust (component: Fe, Si, Al, Ni),Alnico (component: Fe, Al, Ni, Co), a hydrogen absorbing metal (such asa lanthanum nickel alloy (component: La, Ni)), a Co—Cr based alloy, aSnO₂ based oxide, an Nb—Ti alloy, a damping alloy (such as an alloymaterial which reduces and absorbs vibration, and blocks propagation ofvibration, for example, an Al—Zn super plastic alloy, a silent alloy, ornitinol), a material for an electrode, and a semiconductor material(such as silicon, germanium, or potassium arsenide).

The metal may also be deposited or plated with another metal. Furtherthe metal may have different kinds of metals laminated thereon, or maybe a single metal to retain its shape.

A carrier according to the present invention consists essentially of themetal. The carrier may consist of the metal only, or may have the metallaminated on a non-metal material by adhesion, deposition, plating, orthe like.

The shape of the aforementioned carrier is not particularly limited, andexamples of the shape include those of foil, plate, wafer, filter, andbead. Furthermore, it may be the shape of microtiter plate. In order tofacilitate preservation of the obtained result, a back surface of plateor the like may be, for example, applied or coated with a materialusable as a seal or the like (adhesives etc.) so that the material canbe used as a seal.

A solution of a biomolecule is spotted on predetermined positions of theaforementioned carrier. Examples of the biomolecule include nucleicacids, proteins, saccharides, antigens, antibodies, peptides, andenzymes. Hereafter, explanation will be made by exemplifying a nucleicacid as the biomolecule. However, methods and conditions conventionallyused for immobilization can be used also for other substances exceptthat an ultraviolet ray is applied for the immobilization.

The nucleic acid is not particularly different from a usual solidphase-immobilized nucleic acid used for hybridization of nucleic acidsusing a solid phase-immobilized nucleic acid, and it is not particularlylimited so long as it is a nucleic acid that allows hybridization. Forexample, it may be a naturally occurring or synthesized DNA (includingoligonucleotides) or RNA (including oligonucleotides). Further, thenucleic acid may be single-stranded or double-stranded. The chain lengthof the nucleic acid is not also particularly limited so long as itallows hybridization. However, it is usually about 5 to 50,000nucleotides, preferably 20 to 10,000 nucleotides. Furthermore, thenucleic acid may have a polymer of oligonucleotides having a group thatbecomes reactive upon ultraviolet ray irradiation such as those ofthymidine or the like at the 5′ end or 3′ end.

The solvent for dissolving the nucleic acid is not particularly limited,and examples thereof include: distilled water; buffers usually used forpreparing a nucleic acid solution, for example, a Tris buffer such as TEbuffer (10 mM Tris/hydrochloric acid, pH 8.0, 1 mM EDTA); an aqueoussolution containing sodium chloride; an aqueous solution containing acarboxylic acid salt (sodium citrate, ammonium citrate, sodium acetateetc.); an aqueous solution containing a sulfonic acid salt (sodiumdodecylsulfate, ammonium dodecylsulfate etc.); and an aqueous solutioncontaining a phosphonic acid salt (sodium phosphate, ammonium phosphateetc.). Examples thereof further include commercially available solventssuch as Micro Spotting Solution (TeleCHem International, Inc.). Althoughthe concentration of the nucleic acid solution is not particularlylimited either, it is usually a concentration of 1 mmol/ml to 1 fmol/ml,preferably 100 pmol/ml to 100 fmol/ml.

Examples of the method of spotting of the nucleic acid solution on thecarrier include a method involving dropping the nucleic acid solutiononto the carrier with a pipette and a method involving using acommercially available spotter. Although the shape of spot and amount ofthe solution to be spotted are not particularly limited so long as theposition at which the nucleic acid solution has been spotted can beconfirmed, the shape is preferably a dot shape or circular shape. Theamount of the solution to be spotted is preferably 10 nl to 10 ml. Thenucleic acid solution is spotted on one place or two or more places onthe carrier. One kind of nucleic acid solution or two or more kinds ofnucleic acid solutions may be spotted. A labeled nucleic acid may beimmobilized as a positive control indicating immobilization of thenucleic acid on the carrier.

In a preferred embodiment of the present invention, after the nucleicacid solution is spotted on the carrier, an ultraviolet ray containing acomponent having a wavelength of 280 nm is applied. An ultraviolet raycontaining a component having a wavelength of 220 to 300 nm can be givenas the ultraviolet ray. The nucleic acid solution can be dried after thespotting and before the ultraviolet ray irradiation. The nucleic acidsolution may be spontaneously dried, or dried by heating. When it isheated, the heating temperature is usually 30 to 100° C., and preferably35 to 45° C.

Then, an ultraviolet ray containing a component having a wavelength of280 nm is applied at least on the position or positions of the carrierat which the nucleic acid has been immobilized. Specifically, theultraviolet ray may be monochromatic light having a wavelength of 280nm, or an ultraviolet ray having a broad waveform and containing acomponent having a wavelength of 280 nm. Examples of the ultraviolet rayhaving a broad waveform and containing a component having a wavelengthof 280 nm include an ultraviolet ray containing a component having awavelength of 220 to 300 nm. Examples of the ultraviolet ray containinga component having a wavelength of 220 to 300 nm include an ultravioletray having a maximum value at near 280 nm. The irradiation dose isusually 100 mJ/cm² or more, preferably 200 mJ/cm² or more, as cumulativeirradiation dose.

By immobilizing a nucleic acid on a carrier as described above, anucleic acid-immobilized carrier is produced. The nucleicacid-immobilized carrier obtained by the method of the present inventioncan be used for, for example, analysis of nucleic acids based onhybridization. Because a nucleic acid immobilized on a carrier by themethod of the present invention hardly detaches from the carrier underthe conditions of usual hybridization, more favorable detectionsensitivity and reproducibility can be obtained than those in the casewhere the irradiation with an ultraviolet ray is not performed. Thehybridization and detection thereof can be performed in the same manneras hybridization utilizing a usual solid phase-immobilized nucleic acid.

Since an inexpensive metallic material is used as a carrier forimmobilizing a nucleic acid in the present invention, the cost can bereduced. Moreover, since the metallic material can be easily formed, itbecomes easy to produce DNA microarrays of various shapes. Further, themetallic material can be stored for a long period of time, and hassuperior storage stability. Furthermore, the method of the presentinvention does not require a step of coating a surface of carrier, andthus it becomes possible to immobilize nucleic acid directly on a metalused as an electrode or the like. Hybridization of a complementarynucleic acid in the solution and an immobilized nucleic acid can beperformed efficiently by immobilizing a nucleic acid on an electrode.Hybridization is presumably performed efficiently because a nucleicacid, which has negative charge, is attracted to a positive electrodeand then the concentration of the nucleic acid near the positiveelectrode becomes high.

EXAMPLES

Hereafter, the present invention will be explained more specifically byway of examples.

Example 1 Immobilization of Nucleic Acid on Plate

Oligonucleotides having the nucleotide sequences of SEQ ID NOS: 1 and 2respectively (21mer) were synthesized in a conventional manner by usingan oligonucleotide synthesizer (Perkin-elmer Applied Biosystems).Furthermore, DNA having the nucleotide sequence of SEQ ID NO: 3 (262 bp)was also prepared as a probe. The oligonucleotide having the nucleotidesequence of SEQ ID NO: 1 and the probe were biotinylated at the 5′ ends.The oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 wascomplementary to the biotinylated probe. Those oligonucleotides weredissolved in 1×TE buffer (10 mM Tris-HCl, pH 8/1 mM EDTA) at aconcentration of 1 pmol/μl.

Each of the aforementioned oligonucleotide solutions was spotted on acommercially available aluminum foil (Mitsubishi Aluminum Co., Ltd.) asthree spots at predetermined positions (FIG. 1). The amount of thesolutions used for each spotting was 0.5 μl, and the size of each of thespots was about 1 mm in diameter. This aluminum foil was put into adrier and dried at 37° C. for 20 minutes. Then, the foil was irradiatedwith an ultraviolet ray containing a component having a wavelength of280 nm for 250 mJ/cm² by using Uvstratalinker 2400 (STRATAGENE) at adistance of 16 cm. The irradiation time was 100 seconds. Then, thealuminum foil was washed by shaking in water for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the aluminum foil in a similar manner.

Comparative Example 1

The aluminum foil was irradiated beforehand with an ultraviolet raycontaining a component having a wavelength of 280 nm for 250 mJ/cm² byusing Uvstratalinker 2400 (STRATAGENE) at a distance of 16 cm. Each ofthe oligonucleotide solutions described in Example 1 was spotted on thealuminum foil as three spots at predetermined positions. The amount ofthe solutions used for each spotting was 0.5 μl, and the size of each ofthe spots was about 1 mm in diameter. The irradiation time was 100seconds. This aluminum foil was put into a drier and dried at 37° C. for20 minutes. Then, the aluminum foil was washed by shaking in water for30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the aluminum foil in a similar manner.

Example 2 Hybridization and Detection Thereof

(1) Hybridization

On the nucleic acid-immobilized portions of theoligonucleotide-immobilized aluminum foil of Example 1 and ComparativeExample 1, 60 μl of a hybridization solution (Arrayit UniHyb, (TeleCHemInternational, Inc.) containing 3 pmol of the biotinylated probe (262bp) was placed, and the aluminum foil was put into a case shielded fromwater (HybriCassette), immersed in a water bath with the case, andheated at 45° C. for 2 hours.

(2) Post-Hybridization

After the hybridization, post-hybridization washing was performed underthe following conditions to remove the probe non-specifically adsorbedon the oligonucleotide-immobilized aluminum foil.

[Post-Hybridization Washing Conditions]

-   1) 2×SSC, 0.1% SDS; room temperature, 5 minutes, twice-   2) 0.2×SSC, 0.1% SDS; 40° C., 5 minutes, twice-   3) 2×SSC; room temperature, 1 minute, 3 times

(3) Detection of Oligonucleotides Immobilized on Aluminum Foil andHybridization

On the portions of the aluminum foil on which the hybridization solutionwas placed, 1.5 ml of a blocking solution containing milk proteins(BlockAce, Snow Brand Milk Products) was placed to perform blocking atroom temperature for 30 minutes. After the blocking solution wasremoved, 1.5 ml of streptavidin-alkaline phosphatase conjugate solution(VECTOR) was placed and reacted at room temperature for 30 minutes.Then, the aluminum foil was immersed in TBST solution (50 mM Tris-HCl(pH 7.5), 0.15 M NaCl, 0.05% Tween 20) and shaken for 5 minutes toremove the conjugate that did not react. Finally, 1.5 ml of a substratesolution (TMB) was placed on the portions of the aluminum foil on whichthe hybridization solution was placed and left for 30 minutes to performa coloring reaction.

The results are shown in Table 1. The symbols used in Table 1 have thesame meanings as in Table 2 and the other tables mentioned later. Thesignals of the positions at which the oligonucleotide having thesequence of SEQ ID NO: 1 was immobilized indicate amounts of immobilizedoligonucleotides, and the signals of the positions at which theoligonucleotide having the sequence of SEQ ID NO: 2 was immobilizedindicate intensities of hybridization. TABLE 1 Immobilizedoligonucleotide SEQ ID NO: 1 SEQ ID NO: 2 Example 1 ⊚ ⊚ Comparative X XExample 1⊚: Most of signals appeared extremely clearly with extremely highsensitivity.◯: Most of signals appeared clearly with high sensitivity.Δ: A part of signals appeared unclearly or with low sensitivity.X: Most of signals appeared unclearly or with low sensitivity, or nosignal appeared at all.

As apparent from the results shown in Table 1, the oligonucleotides weremore surely immobilized on the oligonucleotide-immobilized aluminum foilof Example 1 than on the oligonucleotide-immobilized aluminum foil ofComparative Example 1. Moreover, on the oligonucleotide-immobilizedaluminum foil of Example 1, the hybridization signals appeared clearly.In addition, on the positions of control (positions at which a solutionnot containing any nucleic acid was spotted), no signal appeared at all.

Example 3 Immobilization of Nucleic Acid on Plate

Oligonucleotides having the nucleotide sequences of SEQ ID NOS: 4, 5,and 6 respectively (31 mer) were synthesized in a conventional manner byusing an oligonucleotide synthesizer (Perkin-elmer Applied Biosystems).The oligonucleotide having the nucleotide sequence of SEQ ID NO: 4 wasbiotinylated at the 5′ end. The oligonucleotides having the nucleotidesequences of SEQ ID NOS: 4 and 5 corresponded to the oligonucleotideshaving the nucleotide sequences of SEQ ID NOS: 1 and 2 described inExample 1 with ten thymidine residues at the 5′ ends, respectively. Theoligonucleotide of SEQ ID NO: 5 was complementary to the aforementionedbiotinylated probe, and the oligonucletide of SEQ ID NO: 6 did not havecomplementarity because it was different from the oligonucleotide of SEQID NO: 5 by one nucleotide. Those oligonucleotides were dissolved in5×SSC at a concentration of 100 pmol/ml.

Each of the aforementioned oligonucleotide solutions was spotted on acommercially available stainless plate (Special Kinzoku Kogyo Co., Ltd.)as three spots at predetermined positions by using a spotter (Pyxsis5500, CARTESIAN). The size of each of the spots was about 0.3 mm indiameter. This plate was put into a drier and dried at 42° C. for 20minutes. Then, the plate was irradiated with an ultraviolet raycontaining a component having a wavelength of 280 nm for 300 mJ/cm² byusing Uvstratalinker 2400 (STRATAGENE) at a distance of 16 cm. Theirradiation time was 120 seconds. Then, the plate was washed by shakingin water for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (2×SSCbuffer) was also subjected to the immobilization operation as a controlby spotting it on the plate in a similar manner.

Comparative Example 2

The stainless plate was irradiated beforehand with an ultraviolet raycontaining a component having a wavelength of 280 nm for 300 mJ/cm² byusing Uvstratalinker 2400 (STRATAGENE) at a distance of 16 cm. Each ofthe oligonucleotide solutions described in Example 3 was spotted on thestainless plate as three spots at predetermined positions by using aspotter (Pyxsis 5500, CARTESIAN). The irradiation time was 120 seconds.This plate was put into a drier and dried at 42° C. for 20 minutes.Then, the plate was washed by shaking in water for 30 minutes, anddried.

On the other hand, a solution not containing any nucleic acid (2×SSCbuffer) was also subjected to the immobilization operation as a controlby spotting it on the plate in a similar manner.

Example 4 Hybridization and Detection Thereof

On the nucleic acid-immobilized portions of theoligonucleotide-immobilized plates of Example 3 and Comparative Example2, 60 ml of a hybridization solution (Arrayit UniHyb, (TeleCHemInternational, Inc.) containing 3 pmol of the biotinylated probe (262bp) was placed, and the plates were put into a case shielded from water(HybriCassette), immersed in a water bath with the case, and heated at45° C. for 2 hours.

Thereafter, post-hybridization, detection of the oligonucleotidesimmobilized on the plates, and hybridization were performed in the samemanner as that of Example 2. The results are shown in Table 2. Thesignals of the positions at which the oligonucleotide having thesequence of SEQ ID NO: 4 was immobilized indicate amounts of immobilizedoligonucleotides, and the signals of the positions at which theoligonucleotide having the sequence of SEQ ID NO: 5 was immobilizedindicate intensities of hybridization. TABLE 2 Immobilizedoligonucleotide SEQ ID SEQ ID SEQ ID NO: 4 NO: 5 NO: 6 Example 3 ⊚ ⊚ XComparative X X X Example 2

As apparent from the results shown in Table 2, the oligonucleotides weremore surely immobilized on the oligonucleotide-immobilized plate ofExample 3 than on the oligonucleotide-immobilized plate of ComparativeExample 2. Moreover, on the oligonucleotide-immobilized plate of Example3, the hybridization signals also appeared clearly. In addition, on thepositions of control (positions at which a solution not containing anynucleic acid was spotted) and SEQ ID NO: 6, no signal appeared at all.

Example 5 Immobilization of Nucleic Acid on Plate

Each of the oligonucleotide solutions prepared in example 3 was spottedon a silver tungsten plate (Eastern Technics Corp.) as three spots atpredetermined positions by using a spotter. The size of each of thespots was about 0.3 mm in diameter. This plate was put into a drier anddried at 42° C. for 20 minutes. Then, the plate was irradiated with anultraviolet ray containing a component having a wavelength of 280 nm for400 mJ/cm² by using Uvstratalinker 2400 (STRATAGENE) at a distance of 16cm. The irradiation time was 160 seconds. Then, the plate was washed byshaking in water for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (2×SSCbuffer) was also subjected to the immobilization operation as a controlby spotting it on the plate in a similar manner.

Comparative Example 3

The silver tungsten plate was irradiated beforehand with an ultravioletray containing a component having a wavelength of 280 nm for 400 mJ/cm²by using Uvstratalinker 2400 (STRATAGENE) at a distance of 16 cm. Eachof the oligonucleotide solutions described in Example 3 was spotted onthe silver tungsten plate as three spots at predetermined positions byusing a spotter (Pyxsis 5500, CARTESIAN). The irradiation time was 160seconds. This plate was put into a drier and dried at 42° C. for 20minutes. Then, the plate was washed by shaking in water for 30 minutes,and dried.

On the other hand, a solution not containing any nucleic acid (2×SSCbuffer) was also subjected to the immobilization operation as a controlby spotting it on the plate in a similar manner.

Example 6 Hybridization and Detection Thereof

On the nucleic acid-immobilized portions of theoligonucleotide-immobilized plates of Example 5 and Comparative Example3, 60 ml of a hybridization solution (Arrayit UniHyb, (TeleCHemInternational, Inc.) containing 3 pmol of the biotinylated probe (262bp) was placed, and the plates were put into a case shielded from water(HybriCassette), immersed in a water bath with the case, and heated at45° C. for 2 hours.

Thereafter, post-hybridization, detection of the oligonucleotidesimmobilized on the plates, and hybridization were performed in the samemanner as that of Example 2. The results are shown in Table 3. TABLE 3Immobilized oligonucleotide SEQ ID SEQ ID SEQ ID NO: 4 NO: 5 NO: 6Example 5 ⊚ ⊚ X Comparative X X X Example 3

As apparent from the results shown in Table 3, the oligonucleotides weremore surely immobilized on the oligonucleotide-immobilized plate ofExample 5 than on the oligonucleotide-immobilized plate of ComparativeExample 3. Moreover, on the oligonucleotide-immobilized plate of Example5, the hybridization signals also appeared clearly. In addition, on thepositions of control (positions at which a solution not containing anynucleic acid was spotted) and SEQ ID NO: 6, no signal appeared at all.

Example 7 Immobilization of Nucleic Acid on Plate

A λ DNA fragment (A) was amplified in a conventional manner by usingoligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 7and 8 as primers. The obtained fragment was subjected to agaroseelectrophoresis and detected by ethidium bromide staining. As a result,it was found that the fragment had a length of about 300b. A λ DNAfragment (B) (about 300b) that was not complementary to theaforementioned λ DNA was also amplified in a similar manner.

A solution of each of the aforementioned λ DNAs was spotted on acommercially available aluminum foil (Mitsubishi Aluminum Co., Ltd.) asthree spots at predetermined positions by using a spotter (Pyxsis 5500,CARTESIAN). The size of each of the spots was about 0.3 mm in diameter.This aluminum foil was put into a drier and dried at 42° C. for 20minutes. Then, the foil was irradiated with an ultraviolet raycontaining a component having a wavelength of 280 nm for 600 mJ/cm² byusing Uvstratalinker 2400 (STRATAGENE) at a distance of 16 cm. Theirradiation time was 240 seconds. Then, the aluminum foil was washed byshaking in water for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the aluminum foil in a similar manner.

Comparative Example 4

Each of the λ DNA solutions described in Example 7 (concentration: 1pmol/μl) was spotted on the aluminum foil as three spots atpredetermined positions by using a spotter (Pyxsis 5500, CARTESIAN).This aluminum foil was put into a drier and dried at 42° C. for 20minutes. Then, the aluminum foil was washed by shaking in water for 30minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the aluminum foil in a similar manner.

Example 8 Hybridization and Detection Thereof

(1) Hybridization

A λ DNA fragment (C) was amplified by using an oligonucleotide havingthe nucleotide sequence shown in SEQ ID NO: 7 labeled with biotin at the5′ end and an oligonucleotide having the nucleotide sequence shown inSEQ ID NO: 8 as primers. The sequence of this λ DNA fragment (C) was thesame as that of λ DNA fragment (A) prepared in Example 7.

The λ DNA-immobilized aluminum foil of each of Example 7 and ComparativeExample 4 was immersed in water heated to 95° C. for 5 minutes, andimmersed in water cooled to 4° C. for 5 minutes. Subsequently, on thenucleic acid-immobilized portions of the λ DNA-immobilized aluminumfoil, 60 ml of a hybridization solution (Arrayit UniHyb, (TeleCHemInternational, Inc.) containing 0.5 pmol of the aforementionedbiotinylated λ DNA fragment (C) was placed, and the aluminum foil wasput into a case shielded from water (HybriCassette), immersed in a waterbath with the case, and heated at 55° C. for 2 hours.

(2) Post-Hybridization

After the hybridization, post-hybridization washing was performed underthe following conditions to remove the probe non-specifically adsorbedon the λ DNA-immobilized aluminum foil.

[Post-Hybridization Washing Conditions]

-   -   1) 2×SSC, 0.1% SDS; room temperature, 5 minutes, twice    -   2) 0.2×SSC, 0.1% SDS; 40° C., 5 minutes, twice    -   3) 2×SSC; room temperature, 1 minute, 3 times

(3) Detection of Hybridization

On the portions of the aluminum foil on which the hybridization solutionwas placed, 1.5 ml of a blocking solution containing milk proteins(BlockAce, Snow Brand Milk Products) was placed to perform blocking atroom temperature for 30 minutes. After the blocking solution wasremoved, 1.5 ml of streptavidin-alkaline phosphatase conjugate solution(VECTOR) was placed and reacted at room temperature for 30 minutes.Then, the aluminum foil was immersed in TBST solution (50 mM Tris-HCl(pH 7.5), 0.15 M NaCl, 0.05% Tween 20) and shaken for 5 minutes toremove the conjugate that did not react. Finally, 1.5 ml of a substrate(TMB) solution was placed on the portions of the aluminum foil on whichthe hybridization solution was placed and left for 30 minutes to performa coloring reaction.

The results are shown in Table 4. TABLE 4 Immobilized nucleic acid λ DNAfragment λ DNA fragment (A) (B) Example 7 ⊚ X Comparative X X Example 4

As apparent from the results shown in Table 4, the λ DNA fragments weresurely immobilized on the λ DNA-immobilized aluminum foil of Example 7,because the hybridization signals specifically and clearly appeared onthe λ DNA-immobilized aluminum foil. On the other hand, on the λDNA-immobilized aluminum foil of Comparative Example 4, no signalappeared at all. In addition, on the positions of control (positions atwhich a solution not containing any nucleic acid was spotted) of the λDNA-immobilized aluminum foil of Example 7 and the λ DNA-immobilizedaluminum foil of Comparative Example 4, no signal appeared at all.

Example 9 Immobilization of Nucleic Acid on Plate

Each of the λ DNA solutions described in Example 7 was spotted on acommercially available stainless plate (Special Kinzoku Kogyo Co., Ltd.)as three spots at predetermined positions by using a spotter (Pyxsis5500, CARTESIAN). The size of each of the spots was about 0.3 mm indiameter. This plate was put into a drier and dried at 42° C. for 20minutes. Then, the plate was irradiated with an ultraviolet raycontaining a component having a wavelength of 280 nm for 1,200 mJ/cm² byusing Uvstratalinker 2400 (STRATAGENE) at a distance of 16 cm. Theirradiation time was 480 seconds. Then, the plate was washed by shakingin water for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the plate in a similar manner.

Comparative Example 5

Each of the λ DNA solutions described in Example 7 was spotted on thestainless plate as three spots at predetermined positions by using aspotter (Pyxsis 5500, CARTESIAN). This plate was put into a drier anddried at 42° C. for 20 minutes. Then, the plate was washed by shaking inwater for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the plate in a similar manner.

Example 10 Hybridization and Detection Thereof

The λ DNA-immobilized plates of Example 9 and Comparative Example 5 wereimmersed in water heated to 95° C. for 10 minutes, and immersed in watercooled to 4° C. for 5 minutes. Subsequently, on the nucleicacid-immobilized portions of the λ DNA-immobilized plates, 60 ml of ahybridization solution (Arrayit UniHyb, (TeleCHem International, Inc.)containing 1 pmol of the biotinylated λ DNA (C) described in Example 8was placed, and the plates were put into a case shielded from water(HybriCassette), immersed in a water bath with the case, and heated at60° C. for 2 hours.

Thereafter, post-hybridization and detection of hybridization wereperformed in the same manner as that of Example 8. The results are shownin Table 5. TABLE 5 Immobilized nucleic acid λ DNA fragment λ DNAfragment (A) (B) Example 9 ⊚ X Comparative X X Example 5

As apparent from the results shown in Table 5, the λ DNA fragments weresurely immobilized on the λ DNA-immobilized plate of Example 9, becausethe hybridization signals specifically and clearly appeared on theplate. On the other hand, no signal appeared at all on the λDNA-immobilized plate of Comparative Example 5. In addition, also on thepositions of control (positions at which a solution not containing anynucleic acid was spotted) of the λ DNA-immobilized plate of Example 9and the λ DNA-immobilized plate of Comparative Example 5, no signalappeared at all.

Example 11 Immobilization of Nucleic Acid on Plate

Each of the λ DNA solutions described in Example 7 was spotted on acommercially available silicon wafer (Mitsubishi Sumitomo Silicon Corp.)as three spots at predetermined positions by using a spotter (Pyxsis5500, CARTESIAN). The size of each of the spots was about 0.3 mm indiameter. This silicon wafer was put into a drier and dried at 42° C.for 20 minutes. Then, the silicon wafer was irradiated with anultraviolet ray containing a component having a wavelength of 280 nm for1,200 mJ/cm² by using Uvstratalinker 2400 (STRATAGENE) at a distance of16 cm. The irradiation time was 480 seconds. Then, the silicon wafer waswashed by shaking in water for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the silicon wafer in a similar manner.

Comparative Example 6

Each of the λ DNA solutions (concentration: 1 pmol/μl) described inExample 7 was spotted on the silicon wafer as three spots atpredetermined positions by using a spotter (Pyxsis 5500, CARTESIAN).This silicon wafer was put into a drier and dried at 42° C. for 20minutes. Then, the silicon wafer was washed by shaking in water for 30minutes, and dried.

Example 12 Hybridization and Detection Thereof

The λ DNA-immobilized silicon wafer of each of Example 11 andComparative Example 6 was immersed in water heated to 95° C. for 10minutes, and immersed in water cooled to 4° C. for 5 minutes.Subsequently, on the nucleic acid-immobilized portions of the λDNA-immobilized silicon wafer, 60 ml of a hybridization solution(Arrayit UniHyb, (TeleCHem International, Inc.) containing 1 pmol of thebiotinylated λ DNA (C) described in Example 8 was placed, and thesilicon wafer was put into a case shielded from water (HybriCassette),immersed in a water bath with the case, and heated at 60° C. for 2hours.

Thereafter, post-hybridization and detection of hybridization wereperformed in the same manner as that of Example 8. The results are shownin Table 6. TABLE 6 Immobilized nucleic acid λ DNA fragment λ DNAfragment (A) (B) Example 11 ⊚ X Comparative X X Example 6

As apparent from the results shown in Table 6, the λ DNA fragments weresurely immobilized on the λ DNA-immobilized silicon wafer of Example 11,because the hybridization signals specifically and clearly appeared onthe silicon wafer. On the other hand, no signal appeared at all on the λDNA-immobilized silicon wafer of Comparative Example 6. In addition,also on the positions of control (positions at which a solution notcontaining any nucleic acid was spotted) of the λ DNA-immobilizedsilicon wafer of Example 11 and the λ DNA-immobilized silicon wafer ofComparative Example 6, no signal appeared at all.

Example 13 Immobilization of Nucleic Acid on Plate

Each of the λ DNA solutions described in Example 7 was spotted on asubstrate obtained by subjecting a glass plate to gold evaporation asthree spots at predetermined positions by using a spotter (Pyxsis 5500,CARTESIAN). The size of each of the spots was about 0.3 mm in diameter.This gold-evaporated glass substrate was put into a drier and dried at42° C. for 20 minutes. Then, the substrate was irradiated with anultraviolet ray containing a component having a wavelength of 280 nm for1,200 mJ/cm² by using Uvstratalinker 2400 (STRATAGENE) at a distance of16 cm. The irradiation time was 480 seconds. Then, the gold-evaporatedglass substrate was washed by shaking in water for 30 minutes, anddried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the gold-evaporated glass substrate in a similarmanner.

Comparative Example 7

Each of the λ DNA solutions (concentration: 1 pmol/μl) described inExample 7 was spotted on the gold-evaporated glass substrate as threespots at predetermined positions by using a spotter (Pyxsis 5500,CARTESIAN). This plate was put into a drier and dried at 42° C. for 20minutes. Then, the gold-evaporated glass substrate was washed by shakingin water for 30 minutes, and dried.

Example 14 Hybridization and Detection Thereof

The λ DNA-immobilized gold-evaporated glass substrate of each of Example13 and Comparative Example 7 was immersed in water heated to 95° C. for10 minutes, and immersed in water cooled to 4° C. for 5 minutes.Subsequently, on the nucleic acid-immobilized portions of the λDNA-immobilized gold-evaporated glass substrate, 60 ml of ahybridization solution (Arrayit UniHyb, (TeleCHem International, Inc.)containing 1 pmol of the biotinylated λ DNA (C) described in Example 8was placed, and the gold-evaporated glass substrate was put into a caseshielded from water (HybriCassette), immersed in a water bath with thecase, and heated at 60° C. for 2 hours.

Thereafter, post-hybridization and detection of hybridization wereperformed in the same manner as that of Example 8. The results are shownin Table 7. TABLE 7 Immobilized nucleic acid λ DNA fragment λ DNAfragment (A) (B) Example 13 ⊚ X Comparative X X Example 7

As apparent from the results shown in Table 7, the λ DNA fragments weresurely immobilized on the λ DNA-immobilized gold-evaporated glasssubstrate of Example 13, because the hybridization signals specificallyand clearly appeared on the gold-evaporated glass substrate. On theother hand, no signal appeared at all on the λ DNA-immobilizedgold-evaporated glass substrate of Comparative Example 7. In addition,also on the positions of control (positions at which a solution notcontaining any nucleic acid was spotted) of the λ DNA-immobilizedgold-evaporated glass substrate of Example 13 and the λ DNA-immobilizedgold-evaporated glass substrate of Comparative Example 7, no signalappeared at all.

Example 15 Immobilization of Nucleic Acid on Plate

Each of the λ DNA solutions described in Example 7 was spotted on acommercially available copper foil (Nikko Metal Manufacturing Co., Ltd)as three spots at predetermined positions by using a spotter (Pyxsis5500, CARTESIAN). The size of each of the spots was about 0.3 mm indiameter. This copper foil was put into a drier and dried at 42° C. for20 minutes. Then, the foil was irradiated with an ultraviolet raycontaining a component having a wavelength of 254 nm for 1,200 mJ/cm² byusing Uvstratalinker 2400 (STRATAGENE) at a distance of 16 cm. Theirradiation time was 480 seconds. Then, the copper foil was washed byshaking in water for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the copper foil in a similar manner.

Comparative Example 8

Each of the λ DNA solutions (concentration: 1 pmol/μl) described inExample 7 was spotted on the copper foil as three spots at predeterminedpositions by using a spotter (Pyxsis 5500, CARTESIAN). This copper foilwas put into a drier and dried at 42° C. for 20 minutes. Then, thecopper foil was washed by shaking in water for 30 minutes, and dried.

Example 16 Hybridization and Detection Thereof

The λ DNA-immobilized copper foil of each of Example 15 and ComparativeExample 8 was immersed in water heated to 95° C. for 10 minutes, andimmersed in water cooled to 4° C. for 5 minutes. Subsequently, on thenucleic acid-immobilized portions of the λ DNA-immobilized copper foil,60 ml of a hybridization solution (Arrayit UniHyb, (TeleCHemInternational, Inc.) containing 1 pmol of the biotinylated λ DNA (C)described in Example 8 was placed, and the copper foil was put into acase shielded from water (HybriCassette), immersed in a water bath withthe case, and heated at 60° C. for 2 hours.

Thereafter, post-hybridization and detection of hybridization wereperformed in the same manner as that of Example 8. The results are shownin Table 8. TABLE 8 Immobilized nucleic acid λ DNA fragment λ DNAfragment (A) (B) Example 15 ⊚ X Comparative X X Example 8

As apparent from the results shown in Table 8, the λ DNA fragments weresurely immobilized on the λ DNA-immobilized copper foil of Example 15,because the hybridization signals specifically and clearly appeared onthe copper foil. On the other hand, no signal appeared at all on the λDNA-immobilized copper foil of Comparative Example 8. In addition, alsoon the positions of control (positions at which a solution notcontaining any nucleic acid was spotted) of the λ DNA-immobilizedgold-evaporated glass substrate of Example 15 and the λ DNA-immobilizedcopper foil of Comparative Example 8, no signal appeared at all.

Example 17 Immobilization of Nucleic Acid on Plate

Each of the λ DNA solutions described in Example 7 was spotted on acommercially available pure nickel foil (Nikko Metal Manufacturing Co.,Ltd) as three spots at predetermined positions by using a spotter(Pyxsis 5500, CARTESIAN). The size of each of the spots was about 0.3 mmin diameter. This pure nickel foil was put into a drier and dried at 42°C. for 20 minutes. Then, the foil was irradiated with an ultraviolet raycontaining a component having a wavelength of 280 nm for 1,200 mJ/cm² byusing Uvstratalinker 2400 (STRATAGENE) at a distance of 16 cm. Theirradiation time was 480 seconds. Then, the pure nickel foil was washedby shaking in water for 30 minutes, and dried.

On the other hand, a solution not containing any nucleic acid (1×TEbuffer) was also subjected to the immobilization operation as a controlby spotting it on the pure nickel foil in a similar manner.

Comparative Example 9

Each of the λ DNA solutions (concentration: 1 pmol/μl) described inExample 7 was spotted on the pure nickel foil as three spots atpredetermined positions by using a spotter (Pyxsis 5500, CARTESIAN).This pure nickel foil was put into a drier and dried at 42° C. for 20minutes. Then, the pure nickel foil was washed by shaking in water for30 minutes, and dried.

Example 18 Hybridization and Detection Thereof

The λ DNA-immobilized pure nickel foil of each of Example 17 andComparative Example 9 was immersed in water heated to 95° C. for 10minutes, and immersed in water cooled to 4° C. for 5 minutes.Subsequently, on the nucleic acid-immobilized portions of the λDNA-immobilized pure nickel foil, 60 ml of a hybridization solution(Arrayit UniHyb, (TeleCHem International, Inc.) containing 1 pmol of thebiotinylated λ DNA (C) described in Example 8 was placed, and the purenickel foil was put into a case shielded from water (HybriCassette),immersed in a water bath with the case, and heated at 60° C. for 2hours.

Thereafter, post-hybridization and detection of hybridization wereperformed in the same manner as that of Example 8. The results are shownin Table 9. TABLE 9 Immobilized nucleic acid λ DNA fragment λ DNAfragment (A) (B) Example 17 ⊚ X Comparative X X Example 9

As apparent from the results shown in Table 9, the λ DNA fragments weresurely immobilized on the λ DNA-immobilized copper foil of Example 17,because the hybridization signals specifically and clearly appeared onthe pure nickel foil. On the other hand, no signal appeared at all onthe λ DNA-immobilized pure nickel foil of Comparative Example 9. Inaddition, also on the positions of control (positions at which asolution not containing any nucleic acid was spotted) of the λDNA-immobilized pure nickel foil of Example 17 and the λ DNA-immobilizedpure nickel foil of Comparative Example 9, no signal appeared at all.

Example 19

Oligonucleotides having the nucleotide sequences of SEQ ID NOS: 9, 10,and 11 respectively (26mer) were synthesized in a conventional manner byusing an oligonucleotide synthesizer (Perkin-elmer Applied Biosystems).The oligonucleotide having the nucleotide sequence of SEQ ID NO: 9 wasbiotinylated at the 5′ end. The oligonucleotides having the nucleotidesequences of SEQ ID NOS: 9 and 10 corresponded to the oligonucleotideshaving the nucleotide sequences of SEQ ID NO: 1 and 2 described inExample 1 with five thymidine residues at the 5′ ends, respectively. Theoligonucleotide of SEQ ID NO: 11 did not have complementarity because itwas different from the oligonucleotide of SEQ ID NO: 5 by onenucleotide. In other words, those oligonucleotides are theoligonucleotide sequences of SEQ ID NOS: 4, 5, and 6 as described inExample 3 with the number of the thymidine residues at the 5′ endreduced to five.

The above oligonucleotides were immobilized in the same manner as inExample 3 using a commercially available stainless plate (SpecialKinzoku Kogyo Co., Ltd.). Thereafter, post-hybridization, and detectionof the oligonucleotides immobilized on the plates and hybridization wereperformed in the same manner as that of Example 2.

Comparative Example 10

Each oligonucleotide was immobilized on a stainless plate in the samemanner as in Comparative Example 2, except that oligonucleotidesolutions described in Example 19 were used.

Example 20

On the nucleic acid-immobilized portions of theoligonucleotide-immobilized plates of Example 19 and Comparative Example10, 60 ml of a hybridization solution (Arrayit UniHyb, (TeleCHemInternational, Inc.) containing 3 pmol of the biotinylated probe (262bp) was placed, and the plates were put into a case shielded from water(HybriCassette), immersed in a water bath with the case, and heated at45° C. for 2 hours.

Thereafter, post-hybridization, and detection of the oligonucleotidesimmobilized on the plates and hybridization were performed in the samemanner as that of Example 2. The results are shown in Table 10. Thesignals of the positions at which the oligonucleotide having thesequence of SEQ ID NO: 9 was immobilized indicate amounts of immobilizedoligonucleotides, and the signals of the positions at which theoligonucleotide having the sequence of SEQ ID NO: 10 was immobilizedindicate intensities of hybridization. TABLE 10 Immobilizedoligonucleotide SEQ ID SEQ ID SEQ ID NO: 9 NO: 10 NO: 11 Example 19 ⊚ ⊚X Comparative X X X Example 10

INDUSTRIAL APPLICABILITY

According to the method of the present invention, a biomolecule, forexample, a nucleic acid, especially a short chain length nucleic acid,can be conveniently and efficiently immobilized on a metallic carrier.Further, as coating of the carrier surface is unnecessary, a biomoleculemay be directly fixed onto a metallic electrode or the like.

1. A method of immobilizing a biomolecule on a carrier, comprising thesteps of: spotting a solution of the biomolecule on the carrier; andirradiating the carrier spotted with the solution of the biomoleculewith an ultraviolet ray containing a component having a wavelength of280 nm, wherein the carrier is made of a metal.
 2. The method accordingto claim 1, wherein the ultraviolet ray contains a component having awavelength of 220 to 300 nm.
 3. The method according to claim 1, whereinthe metal is a metal selected from Groups I, II, III, IV, V, VI, or VIIof second to seventh periods and transition elements in a periodictable, or an alloy containing any of these metals.
 4. The methodaccording to claim 1, wherein the irradiation dose of the ultravioletray is 100 mJ/cm² or more.
 5. The method according to claim 1, whereinthe biomolecule is selected from a nucleic acid, protein, saccharide,antigen, antibody, peptide, or enzyme.
 6. A method of producing abiomolecule-immobilized carrier in which a biomolecule is immobilized ona carrier, comprising the steps of: spotting a solution of thebiomolecule on the carrier; and irradiating the carrier spotted with thesolution of the biomolecule with an ultraviolet ray containing acomponent having a wavelength of 280 nm to immobilize the biomolecule onthe carrier.
 7. The method according to claim 6, wherein the ultravioletray contains a component having a wavelength of 220 to 300 nm.
 8. Themethod according to claim 6, wherein the biomolecule comprises a nucleicacid, and the nucleic acid-immobilized carrier is used for analysis ofthe nucleic acid by hybridization.